- Email: [email protected]
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Fig. 4. Identification of the Bre1 and Lge1 as Rad6 E3 ligase complex that signals for the methylation of histone H3. Extracts of S. cerevisiae mutants missing one of the approximately 4800 nonessential genes were tested for the presence of Lys4-methylated histone H3 by GPS. One of the mutants lacking this histone modification is Bre1 (row H, lane 5). Arrows at position d10 and h3 indicate empty wells as plate markers.
now developing methods that do not require antibody specific for the given posttranslational modification. Acknowledgments We are grateful to Mark Gerber for critical reading of this manuscript. This work was supported in part by Grants from the American Cancer Society (RP69921801), National Institute of Health (1R01CA089455) and a Mallinckrodt Foundation Award to AS’s laboratory. Work in MJ’s laboratory was supported by the James S. McDonnell Foundation. A.S. is a Scholar of the Leukemia & Lymphoma Society.
[14] Purification of Sir2 Proteins from Yeast By Sunil Gangadharan, Sonja Ghidelli, and Rohinton T. Kamakaka Silencing is characterized as the packaging of entire domains of chromatin into a structure that is transcriptionally repressed in a gene nonspecific manner and this state is stably propagated from a mother to the daughter cell. There are four silenced loci in the yeast Saccharomyces
METHODS IN ENZYMOLOGY, VOL. 377
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cerevisiae: the cryptic mating type loci HML and HMR, telomeres and the rDNA repeats (reviewed in Dhillon and Kamakaka1). Silencing of the mating type genes at HML and HMR requires inactivation centers termed silencers that flank the MAT and MATa genes, respectively. A silencer element is analogous to an enhancer element in that it functions in either orientation, is distance-independent and can act to repress unrelated genes. The HM silencers contain binding sites for ORC, Rap1p, and Abf1p. In addition to the proteins that directly bind the silencer elements, there are other proteins that are also necessary for silencing including histones and the products of four genes SIR1, SIR2, SIR3, and SIR4.2 It is believed that the role of the silencer and the silencer-binding components is to efficiently recruit the Sir proteins to the silenced loci. It is noteworthy that neither histones nor the Sir proteins bind DNA in a sequence specific manner. An important advance in understanding the molecular mechanism of silencing was made by the observation that Sir3p and Sir4p bind the N-terminal domains of histones H3 and H4 in vitro.3,4 Also, these same Sir proteins are associated with the entire length of the silenced domain in vivo5,6 reinforcing the view that these proteins are structural components of the silenced chromatin. The prevailing model therefore is that the silencer bound proteins recruit Sir1p and together these further recruit Sir3p and Sir4p. Sir2p on the other hand has been shown to interact with Sir4p7–9 and is most likely recruited to the silenced loci via Sir4p. Silencing at telomeres shares many similarities with silencing at HML and HMR. Telomeric silencing requires Rap1p and the Sir proteins Sir2p, Sir3p, and Sir4p but not Sir1p.10 The multiple Rap1p binding sites at the telomere act as a silencer and Rap1p probably recruits Sir3p and Sir4p along with Sir2p resulting in a repressed domain. Since all three Sir proteins (Sir2p, Sir3p, and Sir4p) are required for silencing at HML, HMR, and telomeres, it is thought that silencing at these three loci is mediated by similar if not the same protein complexes.11 1
N. Dhillon and R. T. Kamakaka, Curr. Opin. Genet. Dev. 12, 188 (2002). J. Rine and I. Herskowitz, Genetics 116, 9 (1987). 3 A. Hecht, T. Laroche, S. Strahl-Bolsinger, S. M. Gasser, and M. Grunstein, Cell 80, 583 (1995). 4 A. A. Carmen, L. Milne, and M. Grunstein, J. Biol. Chem. 277, 4778 (2002). 5 A. Hecht, S. Strahl-Bolsinger, and M. Grunstein, Nature 383, 92 (1996). 6 S. Strahl-Bolsinger, A. Hecht, K. Luo, and M. Grunstein, Genes. Dev. 11, 83 (1997). 7 D. Moazed, A. Kistler, A. Axelrod, J. Rine, and A. D. Johnson, Proc. Natl. Acad. Sci. USA 94, 2186 (1997). 8 D. Moazed and A. D. Johnson, Cell 86, 667 (1996). 9 S. Ghidelli, D. Donze, N. Dhillon, and R. T. Kamakaka, EMBO J. 20, 4522 (2001). 10 O. M. Aparicio, B. L. Billington, and D. E. Gottschling, Cell 66, 1279 (1991). 11 G. Cuperus, R. Shafaatian, and D. Shore, EMBO J. 19, 2641 (2000). 2
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The 1–2 Mb rDNA locus is the fourth and largest repressed locus in S. cerevisiae.12,13 It consists of approximately 200 copies of the 9.1 kbp rDNA repeat. In vivo analysis suggests that approximately half of the genes in the repeats are transcriptionally repressed at any given time. Several proteins including Sir2p affect repression at rDNA although it is not yet known exactly how Sir2p is recruited to this locus. Sir2p is part of a large molecular weight complex called the RENT-complex (Regulator of nucleolar silencing and telophase exit)14,15 that localizes to the nucleolus and which consists of Sir2p, Cdc14p, Net1p, and Nan1p. Sir2p is the only Sir protein that is necessary for repression at all the silenced loci and is also the only Sir protein with a known enzymatic activity. Sir2p possesses an NADþ-dependent deacetylase activity16,17 that can catalyze an NAD-nicotinamide exchange reaction in the presence of acetylated lysines such as those found in the N-termini of histones18 to deacetylate histones in vitro. Sir2p is also the only Sir proteins to be conserved through evolution.19 Histones in chromatin of eukaryotic cells are reversibly acetylated and lysine residues K9 and K14 of histone H3 and K5, K8, and K16 of histone H4 are acetylated in active chromatin and deacetylated in silenced chromatin. Mutational studies indicate that K16 of histone H4 and K9, K14, and K18 of histone H3 are critically important in silencing.3,20–22 Experiments suggest that Sir2p can specifically deacetylate K9 and K14 of histone H3 and K16 of histone H4.16,23,24 Additionally it has been shown that over-expression of Sir2p promotes global deacetylation of histones in vivo.25 12
J. S. Smith and J. D. Boeke, Genes Dev. 11, 241 (1997). M. Bryk, M. Banerjee, M. Murphy, K. E. Knudsen, D. J. Garfinkel, and M. J. Curcio, Genes Dev. 11, 255 (1997). 14 W. Shou, J. H. Seol, A. Shevchenko, C. Baskerville, D. Moazed, Z. W. Chen, J. Jang, H. Charbonneau, and R. J. Deshaies, Cell 97, 233 (1999). 15 A. F. Straight, W. Shou, G. J. Dowd, C. W. Turck, R. J. Deshaies, A. D. Johnson, and D. Moazed, Cell 97, 245 (1999). 16 S. Imai, C. M. Armstrong, M. Kaeberlein, and L. Guarente, Nature 403, 795 (2000). 17 J. Landry, A. Sutton, S. T. Tafrov, R. C. Heller, J. Stebbins, L. Pillus, and R. Sternglanz, Proc. Natl. Acad. Sci. USA 97, 5807 (2000). 18 K. G. Tanner, J. Landry, R. Sternglanz, and J. M. Denu, Proc. Natl. Acad. Sci. USA 97, 14178 (2000). 19 C. B. Brachmann, J. M. Sherman, S. E. Devine, E. E. Cameron, L. Pillus, and J. D. Boeke, Genes Dev. 9, 2888 (1995). 20 J. S. Thompson, X. Ling, and M. Grunstein, Nature 369, 245 (1994). 21 M. Braunstein, R. E. Sobel, C. D. Allis, B. M. Turner, and J. R. Broach, Mol. Cell. Biol. 16, 4349 (1996). 22 N. Suka, Y. Suka, A. A. Carmen, J. Wu, and M. Grunstein, Mol. Cell 8, 473 (2001). 23 A. Kimura, T. Umehara, and M. Horikoshi, Nat. Genet. 32, 370 (2002). 13
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TABLE I Purification of Sir2p Containing Complexes from Yeast Whole Cell Extracts
Column Yeast extract SP-Sepharose Cobalt affinity Q-Sepharose Peak I Peak II Calmodulin affinity Peak I Peak II
Volume (ml)
Protein Conc. (mg/ml)
Total Protein (mg)
200 120 44
2.5 1.58 0.4
500 189 17.6
11 10
0.18 0.38
1.98 3.8
0.04 0.05
0.3 0.35
7.5 7
Purification and characterization of Sir2p containing-complexes is an important step for identifying the proteins that mediate silencing and is the first step in recapitulating the silenced domain in vitro. The purified Sir2p complexes can be used to carry out direct biochemical analysis such as their interactions with nucleosomal templates. The enzymatic activity of Sir2p in the complex and in particular, the activity of mutant complexes can also be examined using a broad variety of substrates. Two nucleosome-binding complexes with distinct deacetylase activities containing Sir2p have been identified in our laboratory.9 One of the complexes is a large multiprotein complex with an approximate molecular weight of 800 kDa, which contains Sir2p, Sir4p, and other unidentified subunits. This complex does not contain either Sir3p or Net1p. The presence of Sir4p in this complex suggested its involvement in silencing at HML, HMR, and the telomeres. Since Net1p is present in the second Sir2p-containing complex, this most likely represents the RENT complex. Tagging Sir2p for Affinity Purification
We used Sir2p with an affinity tag to purify Sir2p-containing complexes from yeast strains. Strains of yeast derived from W303 that carried a His6HA3 epitope tag or a His6-HA-Calmodulin binding peptide tag (TAP tag) fused to the N-terminus of Sir2p were used. The His6-HA-Calmodulin binding peptide tag is similar to the CHH (Calmodulin binding peptideHA-His6) tag reported previously for the purification of a cyclin-CDK 24 25
N. Suka, K. Luo, and M. Grunstein, Nat. Genet. 32, 378 (2002). M. Braunstein, A. B. Rose, S. G. Holmes, C. D. Allis, and J. R. Broach, Genes Dev. 7, 592 (1993).
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Fig. 1. Analysis of purified Sir2p containing complexes. Indicated column fraction from a Superose 6B gel filtration column were assayed for histone deacetylase activity in the presence and absence of NAD.
complex26 and is based on the tandem affinity purification approach using the original TAP tag.27 The tagged SIR2 gene was expressed under the control of its own promoter at the endogenous locus. Strong over expression of the protein using inducible promoters such as the GAL1 gene promoter is not recommended because we have found that under these conditions multiple complexes are formed presumably by nonspecific and/or unnatural interactions with host proteins. We also observe that maximal expression of proteins is obtained in strains where the gene is integrated in the genome rather than on a plasmid since a significant proportion of cells in a culture can survive without plasmid for significant lengths of time reducing yields and generating anomalous subcomplexes. We used standard DNA procedures to introduce the N-terminal tag inframe with the coding region of SIR2 in a pRS406 vector.28 An appropriate restriction fragment was generated from this plasmid and used to transform a strain (JRY4571) that had a URA3 gene downstream of the SIR2 26
S. Honey, B. L. Schneider, D. M. Schieltz, J. R. Yates, and B. Futcher, Nucleic Acids Res. 29, E24 (2001). 27 G. Rigaut, A. Shevchenko, B. Rutz, M. Wilm, M. Mann, and B. Seraphin, Nat. Biotechnol. 17, 1030 (1999). 28 R. Rothstein, Methods Enzymol. 194, 281 (1991).
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promoter. Colonies in which the URA3 gene was replaced with the tagged SIR2 gene fragment were selected on a 5-FOA counter selection plate. Correct integration of the tagged gene at the appropriate locus was confirmed by PCR and Southern blot analysis. Protein blots with antibodies against Sir2p were used to check for expression of the protein. Protein Blot Analysis of Yeast Extracts
Solutions Extrusion Buffer 50 mM HEPES pH 7.0 150 mM NaCl 0.2 mM PMSF 0.5 mM Benzamidine. Extraction Buffer 50 mM HEPES pH 7.0 150 mM NaCl 0.1% NP-40 20 M Zinc acetate 5 mM 2-mercaptoethanol. TBST buffer 0.8% NaCl 0.02% KCl 0.3% Tris–Cl pH 7.5 0.1% Tween 20. Method Colonies to be screened for expression of the tagged protein are grown as 5 ml liquid YPD cultures overnight at 30 to an A600nm 2. Cells are centrifuged at 2500 rpm for 5 min on a bench top centrifuge. The cell pellets are first washed with 1 ml extrusion buffer, transferred to a 1.5 ml microfuge tube. The pellets are resuspended in 200 l extraction buffer, topped with glass beads and vortexed in 6–8 bursts of 30 s each. The bottom of the tube was perforated with a needle and the tube was placed in another microfuge tube and spun at 2500 rpm for 1 min. To the cell suspension add 50 l sample buffer and boil for 5 min. Spin the sample for 1 min at 13,000 rpm and remove the supernatant to a fresh
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tube. Load 20 l of supernatant on to a SDS Polyacrylamide gel. Following electrophoresis, blot the gel onto Immobilon membranes using standard protein blotting techniques. If this method fails to detect proteins expressed at low levels, a smallscale cobalt-bead affinity-binding step can be introduced since the protein is fused to a poly-Histidine tag. For this step, the lysate obtained after vortexing with glass beads is given a high-speed spin and the supernatant added to 50 l of cobalt-beads (BD Talon-Clonetech) that has been previously equilibrated with the binding buffer (50 mM HEPES pH 7.2, 150 mM NaCl, 0.1% NP-40, 10% Glycerol). The lysate and beads are incu bated for 1 h on a rotator at 4 . The beads are spun down at 2000 rpm for 10 s in a microfuge and the supernatant removed. The beads are washed three times with 1 ml of binding buffer. The supernatant is aspirated out and 40 l of sample buffer are added to the beads and boiled for 3 min. The beads are spun down and the supernatant is loaded on a SDS polyacrylamide gel prior to protein blotting. Genotypes of Yeast Strains
ROY 1515: MATa ade2-1 can1-100 his3-11 leu2-3, 112 trp1-1 ura3-1 GAL 6xHis-3xHA-SIR2 pep4:: TRP1 9xMyc-NET1::LEU2 ROY2511: MATa ade2-1 can1-100 his3-11 leu2-3, 112 trp1-1 ura3-1 GAL 6xHis-HA-CBP-SIR2 JRY3009: MATa ade2-1 can1-100 his3-11 leu2–3, 112 trp1-1 ura3-1 GAL JRY 4571:MATa ade2-1 can1-100 his3-11 leu2-3, 112 trp1-1 ura3-1 GAL sir2:: URA3. Assays to Test the Functionality of the Tagged Protein
Mating Assays Patches of the yeast strain with the tagged Sir2p (ROY 1515 or ROY 2511), wild type strain (JRY 3009) and sir2 strain (JRY 4571) are grown on a YPD plate overnight at 30 . A lawn of either MATa his4 cells or MAT his4 cells are spread on YM plates with 300 l YPD to select for diploids that arise from mating events. The plates are allowed to dry and the yeast patches are replica plated onto the lawns and incubated over night at 30 . Cells carrying a functional Sir2p are capable of mating and therefore grow as diploids on the selective plates. In this assay the growth of strains that had Sir2p tagged at the N-terminus with both His6-HA3 epitope tag and CBP-HA-His6 tag was comparable to that of the wild type strain.
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Fig. 2. Histone deacetylase activity of recombinant yeast Sir2p. (A) Effect of trichostatinA on NAD-dependent histone deacetylase activity of E. coli expressed recombinant yeast Sir2p. (B) Effect of CoumermycinA1 on NAD-dependent histone deacetylase activity of E. coli expressed recombinant yeast Sir2p.
Deacetylase Assays Chicken erythrocyte histones were acetylated with recombinant yeast Hat1p acetylase and the acetylated histones were purified as described.29 Histone deacetylase activity was determined using 1 g of acetylated histones with varying amounts of enzyme complex in a final volume of 200 l. The reaction contained 50 mM Tris–HCl pH 9.0, 4 mM MgCl2, 0.2 mM DTT with or without 2 mM NAD. The acetylated histones were added last to the reaction mix. The reaction was incubated for 30 min at 30 and the reaction was stopped by the addition of 50 l of 0.16 M Acetic acid, 0.1 M HCl. The samples were then extracted by the addition of 600 l of ethyl acetate, vortexed and left to stand on the bench for 5 min. Five hundred l of the upper organic phase was removed and diluted with 5 ml scintillation fluid and counted for free acetate. Various inhibitors can also be added into this reaction. Sir2p mediated deacetylation is resistant to 33 nM TrichostatinA but is sensitive to 10 M CoumermycinA1. 29
P. A. Wade, P. L. Jones, D. Vermaak, and A. P. Wolffe, Methods Enzymol. 304, 715 (1999).
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General Considerations While Handling Proteins
All steps are carried out at 4 . Milli-Q water is used to make all solutions. All solutions are filtered using 0.45 m filters. Utmost care has to be taken to reduce keratin contamination and therefore gloves are worn while performing these purification experiments. The purified fractions are stored at 80 after flash freezing in liquid nitrogen. Care should also be taken to reduce foaming of the protein solution. If the complex is to be analyzed for mass spectrometry the buffering ion should be Tris–HCl and glycerol is not recommended in the buffer. Also the concentration of detergent should be as low as possible (0.02%NP40). 1000 Protease Inhibitor Stocks 0.5 M Benzamidine 0.2 M PMSF 100 mg/ml Bacitracin 0.5 M Sodium metabisulphite 5 mg/ml Pepstatin 5 mg/ml TPCK 5 mg/ml TLCK 5 mg/ml Aprotinin 10 mg/ml Leupeptin These are diluted thousand times to get the final concentrations for solutions. Preparation of Yeast Whole Cell Extracts
Solutions Extrusion Buffer 50 mM HEPES pH 7.0 150 mM NaCl All protease inhibitors. Extraction Buffer 50 mM HEPES pH 7.0 150 mM NaCl 0.1% NP-40 20 M Zinc acetate 5 mM 2-mercaptoethanol All protease inhibitors.
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Materials Coffee grinder 10 ml Plastic pipettes Dewar flask Dry ice. Method Inoculate 50 ml of YPD (yeast extract 1%, peptone 2%, dextrose 2%) medium with the yeast strain (ROY1515 or ROY2511) and grow overnight at 30 . with vigorous shaking. This overnight culture is used to inoculate 10 L of YPD and the yeast cell culture is grown to an A600 nm ¼ 11.5. The medium has to be supplemented with adenine if the strain used is defective in adenine biosynthesis. We use 10 two-liter baffle flasks for the growth of the culture though a fermenter can also be used. We typically obtain about 10 g (wet weight) of yeast cells per liter. The cells are pelleted by centrifugation at 5000 rpm for 5 min at 4 using a Sorvall GS-3 rotor. They are washed with 2 L of 50 mM HEPES pH 7.0 and finally the cells are washed with 700 ml extrusion buffer. This is followed by a second wash with 200 ml extrusion buffer. Finally 5 ml of the extrusion buffer is added to the pellet and the cells resuspended as thick slurry with the aid of a glass rod. The thick cell slurry is then added drop-wise into liquid nitrogen in a Dewar flask using a pipette. The frozen cells are removed from the liquid nitrogen and may be stored at 80 . The frozen cells are lysed in a coffee grinder, Braun-Model KSM2household type by the method described.30 Dry ice is added so as to just cover the blades. Approximately 20 g of the frozen cells are ground with constant shaking for 5 min in the grinder. The finely ground frozen powder is transferred to a plastic container but can also be stored at 80 at this stage. To prepare the extract the frozen powder should be thawed on ice in extraction buffer. The choice of extraction buffer depends on the solubility of the protein. We arrived at the extraction conditions for Sir2p by a systematic analysis where isolated yeast nuclei were extracted with varying amounts of salt from 0.05 to 2.0 M NaCl and with different detergents such as NP40, Tween 20, Tween 40, and Triton X-100. The best conditions for extraction were 0.15–0.5 M NaCl plus 0.5% Tween 20. Higher salt
30
M. C. Schultz, D. J. Hockman, T. A. Harkness, W. I. Garinther, and B. A. Altheim, Proc. Natl. Acad. Sci. USA 94, 9034 (1997).
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concentrations with detergent resulted in some of the protein being precipitated out. Varying pH of the buffers can also be tested although we used physiological conditions for pH as this would most likely preserve the complex in its native state. Zinc acetate was included in the buffers as analysis of the primary sequence of Sir2p reveals a potential zinc binding domain which was shown to be required for Sir2p function and which may be required for complex stability. The frozen cells are placed in a beaker and thawed by the addition of 200 ml of extraction buffer containing all of the protease inhibitors. Once the cells have thawed in the extraction buffer, the lysate is mixed and passed through a Yamato homogenizer 1000 rpm for at least 5 times to ensure complete lysis of cells. The homogenate is then cleared from the cell debris by centrifugation at 10,000 rpm for 15 min at 4 in a GS-3 rotor. We consistently get 350 ml of extract with a protein concentration of 2–3 mg/ml. SP-Sepharose FF Ion Exchange Chromatography
The yeast extract is subjected to an initial separation on an ion exchange column. Ion exchange chromatography is capable of separating molecules that have only small differences in charge. This technique is most suited for intermediate steps in purification to collect fractions in a concentrated and semipurified form. SP-sepharose Fast Flow is a strong cation exchanger with excellent flow properties and a high capacity for proteins of almost all pI values. The ion exchange group is a sulphopropyl group that remains charged and maintains consistently high capacities for binding over a pH range of 4–13. Solutions Buffer A 50 mM HEPES pH 7.2 12.5 mM MgCl2 10% Glycerol 0.1% NP-40 All protease inhibitors. Buffer B 50 mM HEPES pH 7.2 12.5 mM MgCl2 10% Glycerol 0.1% NP-40
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1 M KCl All protease inhibitors. Wash Buffer 50 mM HEPES pH 7.4 12.5 mM MgCl2 1 M KCl All protease inhibitors. Materials SP-Sepharose FF (Amersham Pharmacia) XK-26 column (Amersham Pharmacia). Method It is important to empirically determine the binding conditions of the protein to the column such as ionic strength, temperature, pH, and requirement for divalent cations. We chose a pH within the physiological range so that the Sir2p complexes are not disrupted. Glycerol reduces the dielectric constant of the medium and helps to stabilize weak protein-protein interactions. A detergent like NP-40 minimizes nonspecific adsorption of proteins to plastic and glass. The ionic strength was maintained with KCl although NaCl could also be used. Chelating agents such as EGTA and EDTA are left out as they might affect complex stability. Seventy-five milliliters of SP-Sepharose FF beads stored in 20% ethanol, 0.2% sodium acetate were gently washed using a sintered glass funnel in wash buffer (5–7 volumes). The resin was finally resuspended as a 50% (v/v)-slurry in the buffer. One hundred and fifty milliliters of the slurry is enough to pack an XK-26 column of dimensions, 2.6 14.5 cm.2 The column is packed by FPLC using five column volumes of buffer B at a flow rate of 6 ml/min. The column is finally equilibrated with buffer A containing 150 mM KCl. All protein purification was performed with pre-chilled buffers in the cold room at 4–6 . The yeast extract which usually has an ionic strength of 350 mM, was adjusted to 150 mM KCl by adding an equal volume of Buffer A prior to loading on the column. Proteins that do not bind the column during loading are collected. The FPLC is programmed to increase the concentration of KCl in steps of 0.25, 0.40 and 0.6 M. Five column volumes of each buffer concentration are used. The fraction size is approximately one fifth of the volume of the column, which in this case is 15 ml. One hundred microliters of each fraction is collected for Western blot analysis with an anti-HA monoclonal antibody HA.11 (Babco) and protein estimation.
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The Sir2p-containing fractions appear in the 0.4 M KCl elution and peak fractions are pooled and used for the subsequent purification step. Save 100 l of the pooled fractions for protein estimation and Western analysis to determine fold purification. Immobilized Metal Affinity Chromatography (IMAC)
IMAC is a group specific-affinity technique based on the reversible interaction between various amino acid side chains such as histidine, and cysteine with immobilized metal ions such as Ni(II), Co(II) or Zn(II). BD Talon‘ resin is a cobalt based IMAC resin. The metal ion Co(II) is held by a chelator covalently attached to a sepharose CL-6B support. We prefer to use this resin over nickel based IMAC resins as there is significantly less adsorption of unwanted proteins with exposed histidine residues. Another advantage with using this resin is that the bound proteins can be eluted under milder conditions of pH and lower concentration of imidazole. Solutions Binding Buffer A 50 mM HEPES pH 7.2 150 mM NaCl 0.1% NP-40 10% Glycerol All protease inhibitors. Wash Buffer B 50 mM HEPES pH 7.2 150 mM NaCl 0.1% NP-40 10% Glycerol 10 mM Imidazole All protease inhibitors. Elution Buffer C 50 mM HEPES pH 7.2 150 mM NaCl 0.1% NP-40 10% Glycerol 60 mM Imidazole All protease inhibitors.
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Materials 20 ml of BD Talon‘ resin (Clonetech) Econo-pac disposable chromatography column (Biorad). Method Equilibrate 20 ml of the BD Talon‘ resin with buffer A in a 50 ml falcon tube by resuspending in 25 ml of Buffer A, spinning briefly at 1900 rpm and discarding the supernatant. This procedure is repeated four times. A Biorad Econopac (20 ml) disposable column is attached to a fraction collector adjusted to collect 200 drops per fraction (4 ml each). The pooled fractions from the SP-sepharose column containing Sir2p (0.4 M KCl eluate 60 ml) are diluted with an equal volume of binding buffer A lacking NaCl. Following this dilution, the Sir2p containing sample is added to the equilibrated Talon resin in 50 ml falcon tubes. The tubes are rotated for 1 h at 4 , spun briefly at 1900 rpm to collect the supernatant. The supernatant is the flow through for this column and should be saved for analysis. To the resin add an equal volume of wash buffer and pour this slurry into a disposable Biorad Econopac column. Connect a reservoir of buffer B to the column and wash the column with 200 ml of Wash Buffer B (10 column volumes); collecting all fractions. Switch the reservoir from the wash buffer to the elution buffer and continue to collect fractions. Analyze 20 l of all collected fractions by Western blot. Sir2p is typically found in fractions 4–14 of the eluate and these are pooled and used for further chromatographic separation on a Q-sepharose column. Save 100 l of the pooled fractions for protein estimation and Western analysis to determine fold purification. Q-Sepharose Anion Exchange Chromatography
Q-sepharose Fast Flow is an anion exchanger and the exchange group is a quaternary amine that remains charged over a wide range of pH. Solutions Buffer A 50 mM HEPES pH 7.4 12.5 mM MgCl2 0.1% NP-40 10% Glycerol All protease inhibitors.
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Buffer B 1 M KCl 50 mM HEPES pH 7.4 12.5 mM MgCl2 0.1% NP-40 10% Glycerol All protease inhibitors. Materials Q-sepharose FF (Amersham Pharmacia) HR-16 column (Amersham Pharmacia). Method An 8-ml HR16 Q-sepharose column packed on the FPLC is used for the next step of purification of Sir2p. The column is equilibrated with 40 ml of buffer with 0.08 M KCl with a flow rate not to exceed 300 cm/h. The pooled samples from the cobalt column affinity step are diluted with buffer A so that the ionic strength is equivalent to 80 mM KCl. This sample is loaded on to an 8-ml Q-sepharose column and samples are collected as outlined above. The FPLC is programmed to increase the concentration of KCl in steps of 0.08, 0.15, 0.30, and 0.6 M and five column volumes of each buffer concentration are used to collect 2 ml fractions. Fifty microliters of each fraction are removed for protein blot analysis with the anti-HA monoclonal antibody HA.11 and protein estimation. The Sir2p reactive species appears in the 0.15 M KCl fraction and the 0.3 M KCl fractions. The peak fractions from each concentration are pooled and used for the subsequent purification step. Save 50 l of the pooled fractions for protein estimation and Western analysis to determine fold purification. Heparin Sepharose Affinity Chromatography
Heparin sepharose resins are excellent for the purification of DNA binding proteins because they possess properties similar to both ion exchange and affinity resins. Solutions Buffer A 50 mM HEPES pH 7.0 12.5 MgCl2
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10% Glycerol 0.1% NP-40 All protease inhibitors. Materials Heparin-sepharose resin (column volume 2 ml) HR 5/5 column 5 50 mm.2 Method The heparin sepharose resin is packed into a HR 5/5 column and equilibrated in Buffer A with 150 mM KCl. The pooled Sir2p fractions from the Q sepharose column are adjusted to an ionic strength of 150 mM KCl and then loaded onto the equilibrated heparin-sepharose column. The column is washed with three columns of buffer A with 150 mM KCl and a linear gradient of 0.15 to 1 M KCl (10 column volumes) is applied to the column and 0.5 ml fractions collected. The proteins in each fraction are assayed by protein blotting as described above Save 25 l of the pooled fractions for protein estimation and additional Western analysis. Calmodulin Affinity Chromatography
Calmodulin binds with high affinity to a calmodulin-binding peptide in the presence of calcium and removal of calcium from the medium abrogates this interaction. This interaction has been widely and successfully used in the purification of protein complexes where one subunit of the complex contains the calmodulin-binding peptide fused to the coding sequence of the polypeptide.27,31,32 Commercially available calmodulin sepharose beads can be used for the affinity purification of any protein fused to a calmodulin binding peptide tag. Yeast strains containing Sir2p fused to the calmodulin binding peptide were further purified using calmodulin sepharose as a second affinity step and chelation of Ca(II) with EGTA results in the elution of the complex. Care should be taken during this step to determine any change in the size of the protein complex that may occur following removal of divalent cations by EGTA.
31
W. W. Pijnappel, D. Schaft, A. Roguev, A. Shevchenko, H. Tekotte, M. Wilm, G. Rigaut, B. Seraphin, R. Aasland, and A. F. Stewart, Genes Dev. 15, 2991 (2001). 32 A. Roguev, D. Schaft, A. Shevchenko, W. W. Pijnappel, M. Wilm, R. Aasland, and A. F. Stewart, EMBO J. 20, 7137 (2001).
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Solutions Binding Buffer A 50 mM HEPES pH 7.4 150 mM NaCl 10 mM 2-mercaptoethanol 1 mM Magnesium acetate 1 mM Imidazole 2 mM CaCl2 0.1% NP-40 10% Glycerol All protease inhibitors. Elution Buffer B 50 mM HEPES pH 7.4 150 mM NaCl 10 mM 2-mercaptoethanol 1 mM MgOAc 1 mM Imidazole 2 mM EGTA 0.1% NP-40 10% Glycerol All protease inhibitors. Materials Calmodulin Sepharose Econo-pac disposable chromatography column (Biorad). Method Equilibrate 1 ml of calmodulin-sepharose in Buffer A as described for the Talon resin. The pooled Sir2p containing fractions are adjusted for the various buffer components of Buffer A by the addition of components that are missing from the previous step, that is, 10 mM 2-mercaptoethanol, 2 mM CaCl2, and 1 mM MgOAc. This is done by adding an equal volume of Buffer A containing the missing components at twice the concentrations rather than adding the concentrated stocks directly to the Sir2p fractions. The Sir2p-containing fractions are then added to the equilibrated calmodulin sepharose beads and the binding of Sir2p to the beads is allowed to proceed at 4 for 1 h with constant rotation. The beads are then packed into an econo-pac disposable plastic column and washed with 50 ml Binding Buffer A. Elution of the calmodulin bound fraction is carried out using
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Buffer B and 1 ml fractions are collected. Remove 50 l for protein estimation and protein blotting. Gel Filtration Chromatography
Gel filtration chromatography, which is commonly referred to as size exclusion chromatography, is a method for the separation of molecules on the basis of their size and shape. We chose Superose 6 resin because it can withstand higher back-pressure and has low ionic or hydrophobic interactions therefore reducing loss of protein during the purification. The gel filtration chromatography also allows us to change buffers in the sample. We initially checked the apparent molecular weight of Sir2p-containing complexes by applying yeast whole cell lysates, prepared from logarithmically growing cells directly on a Superose 6B column. Column fractions were analyzed by SDS-PAGE followed by immunoblotting using antiserum specific to Sir2p. This analysis indicated that Sir2p eluted from the column in two distinct peaks of apparent molecular weight of 900 and 70 kDa. The 70 kDa is most likely free Sir2p, which has a predicted molecular weight of 63 kDa, while the 900 kDa peak represents the major Sir2p-containing complexes. This analysis was also done initially to determine the stability of the Sir2p complexes under various conditions. A partially purified fraction of the Sir2p-containing complexes was treated with DNAse I, RNAse A, or high concentrations of KCl (500 mM) for 30 min at room temperature and was then subjected to gel filtration analysis. Neither nuclease treatment nor high salt concentration affected the overall size of the complex. Solutions Buffer A 50 mM HEPES 7.4 150 mM KCl 12.5 mM MgCl2 0.1% NP-40 10% Glycerol All the protease inhibitors. Materials Prepacked Superose 6 column (30 ml) HR 10/50 (Amersham Pharmacia).
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Method A Superose 6 column (30 ml size) was equilibrated with 150 ml of buffer A at a flow rate that did not exceed 50 cm/h. Use caution while connecting the column to avoid introducing any air bubbles. Wash the column with 3 volumes of buffer A to equilibrate the resin. Load 2 ml of the Sir2p containing fractions onto the Superose 6 column and collect 1 ml fractions after loading is complete. Remove 100 l of each fraction for protein blotting and Western analysis. These conditions were maintained for all our analysis for ease of comparison across experiments. The Sir2p complex is usually observed in fractions 7–9. It is important to calibrate the gel filtration column by running a set of standard molecular weight marker proteins through the column. Ensure the same volume as sample is also loaded in this case. Red Sepharose Chromatography
Sir2p is an NAD dependant histone deacetylase. We took advantage of this to further purify Sir2p containing complexes using a Red Sepharose CL 6B matrix that has a ligand mimic of NAD, Procion Red HE-3B coupled to it. We used this procedure to further purify and concentrate the sample. Solutions Buffer A 50 mM HEPES pH 7.0 12.5 MgCl2 10% Glycerol 0.1% NP-40 All protease inhibitors. Buffer B 50 mM HEPES pH 7.0 12.5 MgCl2 10% Glycerol 0.1% NP-40 1 M KCl All protease inhibitors. Materials HR 16 column (Amersham Pharmacia).
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Method An 8-ml HR 16 column is packed with the Red Sepharose matrix equilibrated as described for the SP-Sepharose resin. The column is equilibrated in Buffer A with 100 mM KCl. The Sir2p containing fractions are loaded on to this column. The column is then washed with 5 column volumes of 300 mM KCl in buffer A. The bound protein is eluted with 0.1% SDS, 2 ml fractions are collected and analysed by Western blots. We have not been successful in eluting the Sir2p containing complex from this column in its native form. We have also observed that Sir2p binds a Phenyl-Sepharose column with very high affinity in buffer containing 1.0 M KCl but we have once again only been able to elute these complexes from the Phenyl-Sepharose column following denaturation with 0.1% SDS. Purification of Recombinant Sir2p from E. coli
Solutions Lysis Buffer 50 mM HEPES pH 7.0 150 mM NaCl 0.1% Tween 20 20 M Zinc acetate All the protease inhibitors. Wash Buffer 50 mM HEPES pH 7.0 150 mM NaCl 10% Glycerol 10 mM Imidazole HCl All protease inhibitors. Elution Buffer 50 mM HEPES pH 7.0 150 mM NaCl 10% Glycerol 150 mM Imidazole HCl All protease inhibitors. Dialysis Buffer 25 mM HEPES, K+, pH 7.6
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12.5 mM MgCl2 0.1 mM EDTA 10% (v/v) Glycerol 0.1 M KCl 0.1% Tween 20. Materials 20 ml of BD Talon‘ resin (Clonetech) Econo-pac disposable chromatography column (Biorad). Method Transform E. coli DE81 pLysS cells with plasmid pRO 655. The Sir2 coding region is cloned in frame with the T7 6 His tags in a pET28 vector. Select transformants on kanamycin plates. Grow 1 L of cells to an A600 ¼ 0.6 in LB þ Kan. Add IPTG (1 mM final) to induce expression of the recombinant protein and grow the cells for a further 2.5 h. The cells are pelleted by centrifugation at 5000 rpm for 5 min at 4 using a Sorvall GS-3 rotor. The cells are washed in 1 L 20 mM Tris–HCl pH 7.8 and the cell pellets are frozen at 80 . The cell pellets are resuspended in 25 ml of lysis buffer and the A600 of the suspension is measured (10 l in 1 ml water). Sonicate the cell slurry for 2.5 min (30 s 5 pulses). Remeasure A600 (10 l in 1 ml) and the absorbance of the suspension should be approximately 25% of the original. Spin the lysed cell extracts in a Sorval SS34 rotor at 12,000 rpm for 20 min at 4 . The supernatant should contain most of the recombinant Sir2p. The Sir2p containing extract is added to 1.5 ml equilibrated settled BD Talon‘ resin in a 50 ml falcon tube (equilibrated in 50 mM HEPES pH 7.0, 150 mM NaCl, 10% glycerol, 0.5% Tween 20). Rotate the extract for 60 min at 4 on a rotator. Pour this slurry into a disposable Biorad Econopac column attached to a fraction collector. Connect a reservoir of wash buffer to the column and wash the column with 15 ml of Wash Buffer (10 column volumes) collecting fractions. Switch the reservoir from the wash buffer to the elution buffer and continue to collect fractions. Analyze 20 l of all collected fractions by SDS polyacrylamide gels. Dialyze appropriate fractions against dialysis buffer for 3 h at 4 and freeze in aliquots at 80 .
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Fig. 4. Identification of the Bre1 and Lge1 as Rad6 E3 ligase complex that signals for the methylation of histone H3. Extracts of S. cerevisiae mutants missing one of the approximately 4800 nonessential genes were tested for the presence of Lys4-methylated histone H3 by GPS. One of the mutants lacking this histone modification is Bre1 (row H, lane 5). Arrows at position d10 and h3 indicate empty wells as plate markers.
now developing methods that do not require antibody specific for the given posttranslational modification. Acknowledgments We are grateful to Mark Gerber for critical reading of this manuscript. This work was supported in part by Grants from the American Cancer Society (RP69921801), National Institute of Health (1R01CA089455) and a Mallinckrodt Foundation Award to AS’s laboratory. Work in MJ’s laboratory was supported by the James S. McDonnell Foundation. A.S. is a Scholar of the Leukemia & Lymphoma Society.
[14] Purification of Sir2 Proteins from Yeast By Sunil Gangadharan, Sonja Ghidelli, and Rohinton T. Kamakaka Silencing is characterized as the packaging of entire domains of chromatin into a structure that is transcriptionally repressed in a gene nonspecific manner and this state is stably propagated from a mother to the daughter cell. There are four silenced loci in the yeast Saccharomyces
METHODS IN ENZYMOLOGY, VOL. 377
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cerevisiae: the cryptic mating type loci HML and HMR, telomeres and the rDNA repeats (reviewed in Dhillon and Kamakaka1). Silencing of the mating type genes at HML and HMR requires inactivation centers termed silencers that flank the MAT and MATa genes, respectively. A silencer element is analogous to an enhancer element in that it functions in either orientation, is distance-independent and can act to repress unrelated genes. The HM silencers contain binding sites for ORC, Rap1p, and Abf1p. In addition to the proteins that directly bind the silencer elements, there are other proteins that are also necessary for silencing including histones and the products of four genes SIR1, SIR2, SIR3, and SIR4.2 It is believed that the role of the silencer and the silencer-binding components is to efficiently recruit the Sir proteins to the silenced loci. It is noteworthy that neither histones nor the Sir proteins bind DNA in a sequence specific manner. An important advance in understanding the molecular mechanism of silencing was made by the observation that Sir3p and Sir4p bind the N-terminal domains of histones H3 and H4 in vitro.3,4 Also, these same Sir proteins are associated with the entire length of the silenced domain in vivo5,6 reinforcing the view that these proteins are structural components of the silenced chromatin. The prevailing model therefore is that the silencer bound proteins recruit Sir1p and together these further recruit Sir3p and Sir4p. Sir2p on the other hand has been shown to interact with Sir4p7–9 and is most likely recruited to the silenced loci via Sir4p. Silencing at telomeres shares many similarities with silencing at HML and HMR. Telomeric silencing requires Rap1p and the Sir proteins Sir2p, Sir3p, and Sir4p but not Sir1p.10 The multiple Rap1p binding sites at the telomere act as a silencer and Rap1p probably recruits Sir3p and Sir4p along with Sir2p resulting in a repressed domain. Since all three Sir proteins (Sir2p, Sir3p, and Sir4p) are required for silencing at HML, HMR, and telomeres, it is thought that silencing at these three loci is mediated by similar if not the same protein complexes.11 1
N. Dhillon and R. T. Kamakaka, Curr. Opin. Genet. Dev. 12, 188 (2002). J. Rine and I. Herskowitz, Genetics 116, 9 (1987). 3 A. Hecht, T. Laroche, S. Strahl-Bolsinger, S. M. Gasser, and M. Grunstein, Cell 80, 583 (1995). 4 A. A. Carmen, L. Milne, and M. Grunstein, J. Biol. Chem. 277, 4778 (2002). 5 A. Hecht, S. Strahl-Bolsinger, and M. Grunstein, Nature 383, 92 (1996). 6 S. Strahl-Bolsinger, A. Hecht, K. Luo, and M. Grunstein, Genes. Dev. 11, 83 (1997). 7 D. Moazed, A. Kistler, A. Axelrod, J. Rine, and A. D. Johnson, Proc. Natl. Acad. Sci. USA 94, 2186 (1997). 8 D. Moazed and A. D. Johnson, Cell 86, 667 (1996). 9 S. Ghidelli, D. Donze, N. Dhillon, and R. T. Kamakaka, EMBO J. 20, 4522 (2001). 10 O. M. Aparicio, B. L. Billington, and D. E. Gottschling, Cell 66, 1279 (1991). 11 G. Cuperus, R. Shafaatian, and D. Shore, EMBO J. 19, 2641 (2000). 2
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The 1–2 Mb rDNA locus is the fourth and largest repressed locus in S. cerevisiae.12,13 It consists of approximately 200 copies of the 9.1 kbp rDNA repeat. In vivo analysis suggests that approximately half of the genes in the repeats are transcriptionally repressed at any given time. Several proteins including Sir2p affect repression at rDNA although it is not yet known exactly how Sir2p is recruited to this locus. Sir2p is part of a large molecular weight complex called the RENT-complex (Regulator of nucleolar silencing and telophase exit)14,15 that localizes to the nucleolus and which consists of Sir2p, Cdc14p, Net1p, and Nan1p. Sir2p is the only Sir protein that is necessary for repression at all the silenced loci and is also the only Sir protein with a known enzymatic activity. Sir2p possesses an NADþ-dependent deacetylase activity16,17 that can catalyze an NAD-nicotinamide exchange reaction in the presence of acetylated lysines such as those found in the N-termini of histones18 to deacetylate histones in vitro. Sir2p is also the only Sir proteins to be conserved through evolution.19 Histones in chromatin of eukaryotic cells are reversibly acetylated and lysine residues K9 and K14 of histone H3 and K5, K8, and K16 of histone H4 are acetylated in active chromatin and deacetylated in silenced chromatin. Mutational studies indicate that K16 of histone H4 and K9, K14, and K18 of histone H3 are critically important in silencing.3,20–22 Experiments suggest that Sir2p can specifically deacetylate K9 and K14 of histone H3 and K16 of histone H4.16,23,24 Additionally it has been shown that over-expression of Sir2p promotes global deacetylation of histones in vivo.25 12
J. S. Smith and J. D. Boeke, Genes Dev. 11, 241 (1997). M. Bryk, M. Banerjee, M. Murphy, K. E. Knudsen, D. J. Garfinkel, and M. J. Curcio, Genes Dev. 11, 255 (1997). 14 W. Shou, J. H. Seol, A. Shevchenko, C. Baskerville, D. Moazed, Z. W. Chen, J. Jang, H. Charbonneau, and R. J. Deshaies, Cell 97, 233 (1999). 15 A. F. Straight, W. Shou, G. J. Dowd, C. W. Turck, R. J. Deshaies, A. D. Johnson, and D. Moazed, Cell 97, 245 (1999). 16 S. Imai, C. M. Armstrong, M. Kaeberlein, and L. Guarente, Nature 403, 795 (2000). 17 J. Landry, A. Sutton, S. T. Tafrov, R. C. Heller, J. Stebbins, L. Pillus, and R. Sternglanz, Proc. Natl. Acad. Sci. USA 97, 5807 (2000). 18 K. G. Tanner, J. Landry, R. Sternglanz, and J. M. Denu, Proc. Natl. Acad. Sci. USA 97, 14178 (2000). 19 C. B. Brachmann, J. M. Sherman, S. E. Devine, E. E. Cameron, L. Pillus, and J. D. Boeke, Genes Dev. 9, 2888 (1995). 20 J. S. Thompson, X. Ling, and M. Grunstein, Nature 369, 245 (1994). 21 M. Braunstein, R. E. Sobel, C. D. Allis, B. M. Turner, and J. R. Broach, Mol. Cell. Biol. 16, 4349 (1996). 22 N. Suka, Y. Suka, A. A. Carmen, J. Wu, and M. Grunstein, Mol. Cell 8, 473 (2001). 23 A. Kimura, T. Umehara, and M. Horikoshi, Nat. Genet. 32, 370 (2002). 13
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TABLE I Purification of Sir2p Containing Complexes from Yeast Whole Cell Extracts
Column Yeast extract SP-Sepharose Cobalt affinity Q-Sepharose Peak I Peak II Calmodulin affinity Peak I Peak II
Volume (ml)
Protein Conc. (mg/ml)
Total Protein (mg)
200 120 44
2.5 1.58 0.4
500 189 17.6
11 10
0.18 0.38
1.98 3.8
0.04 0.05
0.3 0.35
7.5 7
Purification and characterization of Sir2p containing-complexes is an important step for identifying the proteins that mediate silencing and is the first step in recapitulating the silenced domain in vitro. The purified Sir2p complexes can be used to carry out direct biochemical analysis such as their interactions with nucleosomal templates. The enzymatic activity of Sir2p in the complex and in particular, the activity of mutant complexes can also be examined using a broad variety of substrates. Two nucleosome-binding complexes with distinct deacetylase activities containing Sir2p have been identified in our laboratory.9 One of the complexes is a large multiprotein complex with an approximate molecular weight of 800 kDa, which contains Sir2p, Sir4p, and other unidentified subunits. This complex does not contain either Sir3p or Net1p. The presence of Sir4p in this complex suggested its involvement in silencing at HML, HMR, and the telomeres. Since Net1p is present in the second Sir2p-containing complex, this most likely represents the RENT complex. Tagging Sir2p for Affinity Purification
We used Sir2p with an affinity tag to purify Sir2p-containing complexes from yeast strains. Strains of yeast derived from W303 that carried a His6HA3 epitope tag or a His6-HA-Calmodulin binding peptide tag (TAP tag) fused to the N-terminus of Sir2p were used. The His6-HA-Calmodulin binding peptide tag is similar to the CHH (Calmodulin binding peptideHA-His6) tag reported previously for the purification of a cyclin-CDK 24 25
N. Suka, K. Luo, and M. Grunstein, Nat. Genet. 32, 378 (2002). M. Braunstein, A. B. Rose, S. G. Holmes, C. D. Allis, and J. R. Broach, Genes Dev. 7, 592 (1993).
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Fig. 1. Analysis of purified Sir2p containing complexes. Indicated column fraction from a Superose 6B gel filtration column were assayed for histone deacetylase activity in the presence and absence of NAD.
complex26 and is based on the tandem affinity purification approach using the original TAP tag.27 The tagged SIR2 gene was expressed under the control of its own promoter at the endogenous locus. Strong over expression of the protein using inducible promoters such as the GAL1 gene promoter is not recommended because we have found that under these conditions multiple complexes are formed presumably by nonspecific and/or unnatural interactions with host proteins. We also observe that maximal expression of proteins is obtained in strains where the gene is integrated in the genome rather than on a plasmid since a significant proportion of cells in a culture can survive without plasmid for significant lengths of time reducing yields and generating anomalous subcomplexes. We used standard DNA procedures to introduce the N-terminal tag inframe with the coding region of SIR2 in a pRS406 vector.28 An appropriate restriction fragment was generated from this plasmid and used to transform a strain (JRY4571) that had a URA3 gene downstream of the SIR2 26
S. Honey, B. L. Schneider, D. M. Schieltz, J. R. Yates, and B. Futcher, Nucleic Acids Res. 29, E24 (2001). 27 G. Rigaut, A. Shevchenko, B. Rutz, M. Wilm, M. Mann, and B. Seraphin, Nat. Biotechnol. 17, 1030 (1999). 28 R. Rothstein, Methods Enzymol. 194, 281 (1991).
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promoter. Colonies in which the URA3 gene was replaced with the tagged SIR2 gene fragment were selected on a 5-FOA counter selection plate. Correct integration of the tagged gene at the appropriate locus was confirmed by PCR and Southern blot analysis. Protein blots with antibodies against Sir2p were used to check for expression of the protein. Protein Blot Analysis of Yeast Extracts
Solutions Extrusion Buffer 50 mM HEPES pH 7.0 150 mM NaCl 0.2 mM PMSF 0.5 mM Benzamidine. Extraction Buffer 50 mM HEPES pH 7.0 150 mM NaCl 0.1% NP-40 20 M Zinc acetate 5 mM 2-mercaptoethanol. TBST buffer 0.8% NaCl 0.02% KCl 0.3% Tris–Cl pH 7.5 0.1% Tween 20. Method Colonies to be screened for expression of the tagged protein are grown as 5 ml liquid YPD cultures overnight at 30 to an A600nm 2. Cells are centrifuged at 2500 rpm for 5 min on a bench top centrifuge. The cell pellets are first washed with 1 ml extrusion buffer, transferred to a 1.5 ml microfuge tube. The pellets are resuspended in 200 l extraction buffer, topped with glass beads and vortexed in 6–8 bursts of 30 s each. The bottom of the tube was perforated with a needle and the tube was placed in another microfuge tube and spun at 2500 rpm for 1 min. To the cell suspension add 50 l sample buffer and boil for 5 min. Spin the sample for 1 min at 13,000 rpm and remove the supernatant to a fresh
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tube. Load 20 l of supernatant on to a SDS Polyacrylamide gel. Following electrophoresis, blot the gel onto Immobilon membranes using standard protein blotting techniques. If this method fails to detect proteins expressed at low levels, a smallscale cobalt-bead affinity-binding step can be introduced since the protein is fused to a poly-Histidine tag. For this step, the lysate obtained after vortexing with glass beads is given a high-speed spin and the supernatant added to 50 l of cobalt-beads (BD Talon-Clonetech) that has been previously equilibrated with the binding buffer (50 mM HEPES pH 7.2, 150 mM NaCl, 0.1% NP-40, 10% Glycerol). The lysate and beads are incu bated for 1 h on a rotator at 4 . The beads are spun down at 2000 rpm for 10 s in a microfuge and the supernatant removed. The beads are washed three times with 1 ml of binding buffer. The supernatant is aspirated out and 40 l of sample buffer are added to the beads and boiled for 3 min. The beads are spun down and the supernatant is loaded on a SDS polyacrylamide gel prior to protein blotting. Genotypes of Yeast Strains
ROY 1515: MATa ade2-1 can1-100 his3-11 leu2-3, 112 trp1-1 ura3-1 GAL 6xHis-3xHA-SIR2 pep4:: TRP1 9xMyc-NET1::LEU2 ROY2511: MATa ade2-1 can1-100 his3-11 leu2-3, 112 trp1-1 ura3-1 GAL 6xHis-HA-CBP-SIR2 JRY3009: MATa ade2-1 can1-100 his3-11 leu2–3, 112 trp1-1 ura3-1 GAL JRY 4571:MATa ade2-1 can1-100 his3-11 leu2-3, 112 trp1-1 ura3-1 GAL sir2:: URA3. Assays to Test the Functionality of the Tagged Protein
Mating Assays Patches of the yeast strain with the tagged Sir2p (ROY 1515 or ROY 2511), wild type strain (JRY 3009) and sir2 strain (JRY 4571) are grown on a YPD plate overnight at 30 . A lawn of either MATa his4 cells or MAT his4 cells are spread on YM plates with 300 l YPD to select for diploids that arise from mating events. The plates are allowed to dry and the yeast patches are replica plated onto the lawns and incubated over night at 30 . Cells carrying a functional Sir2p are capable of mating and therefore grow as diploids on the selective plates. In this assay the growth of strains that had Sir2p tagged at the N-terminus with both His6-HA3 epitope tag and CBP-HA-His6 tag was comparable to that of the wild type strain.
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Fig. 2. Histone deacetylase activity of recombinant yeast Sir2p. (A) Effect of trichostatinA on NAD-dependent histone deacetylase activity of E. coli expressed recombinant yeast Sir2p. (B) Effect of CoumermycinA1 on NAD-dependent histone deacetylase activity of E. coli expressed recombinant yeast Sir2p.
Deacetylase Assays Chicken erythrocyte histones were acetylated with recombinant yeast Hat1p acetylase and the acetylated histones were purified as described.29 Histone deacetylase activity was determined using 1 g of acetylated histones with varying amounts of enzyme complex in a final volume of 200 l. The reaction contained 50 mM Tris–HCl pH 9.0, 4 mM MgCl2, 0.2 mM DTT with or without 2 mM NAD. The acetylated histones were added last to the reaction mix. The reaction was incubated for 30 min at 30 and the reaction was stopped by the addition of 50 l of 0.16 M Acetic acid, 0.1 M HCl. The samples were then extracted by the addition of 600 l of ethyl acetate, vortexed and left to stand on the bench for 5 min. Five hundred l of the upper organic phase was removed and diluted with 5 ml scintillation fluid and counted for free acetate. Various inhibitors can also be added into this reaction. Sir2p mediated deacetylation is resistant to 33 nM TrichostatinA but is sensitive to 10 M CoumermycinA1. 29
P. A. Wade, P. L. Jones, D. Vermaak, and A. P. Wolffe, Methods Enzymol. 304, 715 (1999).
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General Considerations While Handling Proteins
All steps are carried out at 4 . Milli-Q water is used to make all solutions. All solutions are filtered using 0.45 m filters. Utmost care has to be taken to reduce keratin contamination and therefore gloves are worn while performing these purification experiments. The purified fractions are stored at 80 after flash freezing in liquid nitrogen. Care should also be taken to reduce foaming of the protein solution. If the complex is to be analyzed for mass spectrometry the buffering ion should be Tris–HCl and glycerol is not recommended in the buffer. Also the concentration of detergent should be as low as possible (0.02%NP40). 1000 Protease Inhibitor Stocks 0.5 M Benzamidine 0.2 M PMSF 100 mg/ml Bacitracin 0.5 M Sodium metabisulphite 5 mg/ml Pepstatin 5 mg/ml TPCK 5 mg/ml TLCK 5 mg/ml Aprotinin 10 mg/ml Leupeptin These are diluted thousand times to get the final concentrations for solutions. Preparation of Yeast Whole Cell Extracts
Solutions Extrusion Buffer 50 mM HEPES pH 7.0 150 mM NaCl All protease inhibitors. Extraction Buffer 50 mM HEPES pH 7.0 150 mM NaCl 0.1% NP-40 20 M Zinc acetate 5 mM 2-mercaptoethanol All protease inhibitors.
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Materials Coffee grinder 10 ml Plastic pipettes Dewar flask Dry ice. Method Inoculate 50 ml of YPD (yeast extract 1%, peptone 2%, dextrose 2%) medium with the yeast strain (ROY1515 or ROY2511) and grow overnight at 30 . with vigorous shaking. This overnight culture is used to inoculate 10 L of YPD and the yeast cell culture is grown to an A600 nm ¼ 11.5. The medium has to be supplemented with adenine if the strain used is defective in adenine biosynthesis. We use 10 two-liter baffle flasks for the growth of the culture though a fermenter can also be used. We typically obtain about 10 g (wet weight) of yeast cells per liter. The cells are pelleted by centrifugation at 5000 rpm for 5 min at 4 using a Sorvall GS-3 rotor. They are washed with 2 L of 50 mM HEPES pH 7.0 and finally the cells are washed with 700 ml extrusion buffer. This is followed by a second wash with 200 ml extrusion buffer. Finally 5 ml of the extrusion buffer is added to the pellet and the cells resuspended as thick slurry with the aid of a glass rod. The thick cell slurry is then added drop-wise into liquid nitrogen in a Dewar flask using a pipette. The frozen cells are removed from the liquid nitrogen and may be stored at 80 . The frozen cells are lysed in a coffee grinder, Braun-Model KSM2household type by the method described.30 Dry ice is added so as to just cover the blades. Approximately 20 g of the frozen cells are ground with constant shaking for 5 min in the grinder. The finely ground frozen powder is transferred to a plastic container but can also be stored at 80 at this stage. To prepare the extract the frozen powder should be thawed on ice in extraction buffer. The choice of extraction buffer depends on the solubility of the protein. We arrived at the extraction conditions for Sir2p by a systematic analysis where isolated yeast nuclei were extracted with varying amounts of salt from 0.05 to 2.0 M NaCl and with different detergents such as NP40, Tween 20, Tween 40, and Triton X-100. The best conditions for extraction were 0.15–0.5 M NaCl plus 0.5% Tween 20. Higher salt
30
M. C. Schultz, D. J. Hockman, T. A. Harkness, W. I. Garinther, and B. A. Altheim, Proc. Natl. Acad. Sci. USA 94, 9034 (1997).
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concentrations with detergent resulted in some of the protein being precipitated out. Varying pH of the buffers can also be tested although we used physiological conditions for pH as this would most likely preserve the complex in its native state. Zinc acetate was included in the buffers as analysis of the primary sequence of Sir2p reveals a potential zinc binding domain which was shown to be required for Sir2p function and which may be required for complex stability. The frozen cells are placed in a beaker and thawed by the addition of 200 ml of extraction buffer containing all of the protease inhibitors. Once the cells have thawed in the extraction buffer, the lysate is mixed and passed through a Yamato homogenizer 1000 rpm for at least 5 times to ensure complete lysis of cells. The homogenate is then cleared from the cell debris by centrifugation at 10,000 rpm for 15 min at 4 in a GS-3 rotor. We consistently get 350 ml of extract with a protein concentration of 2–3 mg/ml. SP-Sepharose FF Ion Exchange Chromatography
The yeast extract is subjected to an initial separation on an ion exchange column. Ion exchange chromatography is capable of separating molecules that have only small differences in charge. This technique is most suited for intermediate steps in purification to collect fractions in a concentrated and semipurified form. SP-sepharose Fast Flow is a strong cation exchanger with excellent flow properties and a high capacity for proteins of almost all pI values. The ion exchange group is a sulphopropyl group that remains charged and maintains consistently high capacities for binding over a pH range of 4–13. Solutions Buffer A 50 mM HEPES pH 7.2 12.5 mM MgCl2 10% Glycerol 0.1% NP-40 All protease inhibitors. Buffer B 50 mM HEPES pH 7.2 12.5 mM MgCl2 10% Glycerol 0.1% NP-40
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1 M KCl All protease inhibitors. Wash Buffer 50 mM HEPES pH 7.4 12.5 mM MgCl2 1 M KCl All protease inhibitors. Materials SP-Sepharose FF (Amersham Pharmacia) XK-26 column (Amersham Pharmacia). Method It is important to empirically determine the binding conditions of the protein to the column such as ionic strength, temperature, pH, and requirement for divalent cations. We chose a pH within the physiological range so that the Sir2p complexes are not disrupted. Glycerol reduces the dielectric constant of the medium and helps to stabilize weak protein-protein interactions. A detergent like NP-40 minimizes nonspecific adsorption of proteins to plastic and glass. The ionic strength was maintained with KCl although NaCl could also be used. Chelating agents such as EGTA and EDTA are left out as they might affect complex stability. Seventy-five milliliters of SP-Sepharose FF beads stored in 20% ethanol, 0.2% sodium acetate were gently washed using a sintered glass funnel in wash buffer (5–7 volumes). The resin was finally resuspended as a 50% (v/v)-slurry in the buffer. One hundred and fifty milliliters of the slurry is enough to pack an XK-26 column of dimensions, 2.6 14.5 cm.2 The column is packed by FPLC using five column volumes of buffer B at a flow rate of 6 ml/min. The column is finally equilibrated with buffer A containing 150 mM KCl. All protein purification was performed with pre-chilled buffers in the cold room at 4–6 . The yeast extract which usually has an ionic strength of 350 mM, was adjusted to 150 mM KCl by adding an equal volume of Buffer A prior to loading on the column. Proteins that do not bind the column during loading are collected. The FPLC is programmed to increase the concentration of KCl in steps of 0.25, 0.40 and 0.6 M. Five column volumes of each buffer concentration are used. The fraction size is approximately one fifth of the volume of the column, which in this case is 15 ml. One hundred microliters of each fraction is collected for Western blot analysis with an anti-HA monoclonal antibody HA.11 (Babco) and protein estimation.
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The Sir2p-containing fractions appear in the 0.4 M KCl elution and peak fractions are pooled and used for the subsequent purification step. Save 100 l of the pooled fractions for protein estimation and Western analysis to determine fold purification. Immobilized Metal Affinity Chromatography (IMAC)
IMAC is a group specific-affinity technique based on the reversible interaction between various amino acid side chains such as histidine, and cysteine with immobilized metal ions such as Ni(II), Co(II) or Zn(II). BD Talon‘ resin is a cobalt based IMAC resin. The metal ion Co(II) is held by a chelator covalently attached to a sepharose CL-6B support. We prefer to use this resin over nickel based IMAC resins as there is significantly less adsorption of unwanted proteins with exposed histidine residues. Another advantage with using this resin is that the bound proteins can be eluted under milder conditions of pH and lower concentration of imidazole. Solutions Binding Buffer A 50 mM HEPES pH 7.2 150 mM NaCl 0.1% NP-40 10% Glycerol All protease inhibitors. Wash Buffer B 50 mM HEPES pH 7.2 150 mM NaCl 0.1% NP-40 10% Glycerol 10 mM Imidazole All protease inhibitors. Elution Buffer C 50 mM HEPES pH 7.2 150 mM NaCl 0.1% NP-40 10% Glycerol 60 mM Imidazole All protease inhibitors.
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Materials 20 ml of BD Talon‘ resin (Clonetech) Econo-pac disposable chromatography column (Biorad). Method Equilibrate 20 ml of the BD Talon‘ resin with buffer A in a 50 ml falcon tube by resuspending in 25 ml of Buffer A, spinning briefly at 1900 rpm and discarding the supernatant. This procedure is repeated four times. A Biorad Econopac (20 ml) disposable column is attached to a fraction collector adjusted to collect 200 drops per fraction (4 ml each). The pooled fractions from the SP-sepharose column containing Sir2p (0.4 M KCl eluate 60 ml) are diluted with an equal volume of binding buffer A lacking NaCl. Following this dilution, the Sir2p containing sample is added to the equilibrated Talon resin in 50 ml falcon tubes. The tubes are rotated for 1 h at 4 , spun briefly at 1900 rpm to collect the supernatant. The supernatant is the flow through for this column and should be saved for analysis. To the resin add an equal volume of wash buffer and pour this slurry into a disposable Biorad Econopac column. Connect a reservoir of buffer B to the column and wash the column with 200 ml of Wash Buffer B (10 column volumes); collecting all fractions. Switch the reservoir from the wash buffer to the elution buffer and continue to collect fractions. Analyze 20 l of all collected fractions by Western blot. Sir2p is typically found in fractions 4–14 of the eluate and these are pooled and used for further chromatographic separation on a Q-sepharose column. Save 100 l of the pooled fractions for protein estimation and Western analysis to determine fold purification. Q-Sepharose Anion Exchange Chromatography
Q-sepharose Fast Flow is an anion exchanger and the exchange group is a quaternary amine that remains charged over a wide range of pH. Solutions Buffer A 50 mM HEPES pH 7.4 12.5 mM MgCl2 0.1% NP-40 10% Glycerol All protease inhibitors.
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Buffer B 1 M KCl 50 mM HEPES pH 7.4 12.5 mM MgCl2 0.1% NP-40 10% Glycerol All protease inhibitors. Materials Q-sepharose FF (Amersham Pharmacia) HR-16 column (Amersham Pharmacia). Method An 8-ml HR16 Q-sepharose column packed on the FPLC is used for the next step of purification of Sir2p. The column is equilibrated with 40 ml of buffer with 0.08 M KCl with a flow rate not to exceed 300 cm/h. The pooled samples from the cobalt column affinity step are diluted with buffer A so that the ionic strength is equivalent to 80 mM KCl. This sample is loaded on to an 8-ml Q-sepharose column and samples are collected as outlined above. The FPLC is programmed to increase the concentration of KCl in steps of 0.08, 0.15, 0.30, and 0.6 M and five column volumes of each buffer concentration are used to collect 2 ml fractions. Fifty microliters of each fraction are removed for protein blot analysis with the anti-HA monoclonal antibody HA.11 and protein estimation. The Sir2p reactive species appears in the 0.15 M KCl fraction and the 0.3 M KCl fractions. The peak fractions from each concentration are pooled and used for the subsequent purification step. Save 50 l of the pooled fractions for protein estimation and Western analysis to determine fold purification. Heparin Sepharose Affinity Chromatography
Heparin sepharose resins are excellent for the purification of DNA binding proteins because they possess properties similar to both ion exchange and affinity resins. Solutions Buffer A 50 mM HEPES pH 7.0 12.5 MgCl2
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10% Glycerol 0.1% NP-40 All protease inhibitors. Materials Heparin-sepharose resin (column volume 2 ml) HR 5/5 column 5 50 mm.2 Method The heparin sepharose resin is packed into a HR 5/5 column and equilibrated in Buffer A with 150 mM KCl. The pooled Sir2p fractions from the Q sepharose column are adjusted to an ionic strength of 150 mM KCl and then loaded onto the equilibrated heparin-sepharose column. The column is washed with three columns of buffer A with 150 mM KCl and a linear gradient of 0.15 to 1 M KCl (10 column volumes) is applied to the column and 0.5 ml fractions collected. The proteins in each fraction are assayed by protein blotting as described above Save 25 l of the pooled fractions for protein estimation and additional Western analysis. Calmodulin Affinity Chromatography
Calmodulin binds with high affinity to a calmodulin-binding peptide in the presence of calcium and removal of calcium from the medium abrogates this interaction. This interaction has been widely and successfully used in the purification of protein complexes where one subunit of the complex contains the calmodulin-binding peptide fused to the coding sequence of the polypeptide.27,31,32 Commercially available calmodulin sepharose beads can be used for the affinity purification of any protein fused to a calmodulin binding peptide tag. Yeast strains containing Sir2p fused to the calmodulin binding peptide were further purified using calmodulin sepharose as a second affinity step and chelation of Ca(II) with EGTA results in the elution of the complex. Care should be taken during this step to determine any change in the size of the protein complex that may occur following removal of divalent cations by EGTA.
31
W. W. Pijnappel, D. Schaft, A. Roguev, A. Shevchenko, H. Tekotte, M. Wilm, G. Rigaut, B. Seraphin, R. Aasland, and A. F. Stewart, Genes Dev. 15, 2991 (2001). 32 A. Roguev, D. Schaft, A. Shevchenko, W. W. Pijnappel, M. Wilm, R. Aasland, and A. F. Stewart, EMBO J. 20, 7137 (2001).
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Solutions Binding Buffer A 50 mM HEPES pH 7.4 150 mM NaCl 10 mM 2-mercaptoethanol 1 mM Magnesium acetate 1 mM Imidazole 2 mM CaCl2 0.1% NP-40 10% Glycerol All protease inhibitors. Elution Buffer B 50 mM HEPES pH 7.4 150 mM NaCl 10 mM 2-mercaptoethanol 1 mM MgOAc 1 mM Imidazole 2 mM EGTA 0.1% NP-40 10% Glycerol All protease inhibitors. Materials Calmodulin Sepharose Econo-pac disposable chromatography column (Biorad). Method Equilibrate 1 ml of calmodulin-sepharose in Buffer A as described for the Talon resin. The pooled Sir2p containing fractions are adjusted for the various buffer components of Buffer A by the addition of components that are missing from the previous step, that is, 10 mM 2-mercaptoethanol, 2 mM CaCl2, and 1 mM MgOAc. This is done by adding an equal volume of Buffer A containing the missing components at twice the concentrations rather than adding the concentrated stocks directly to the Sir2p fractions. The Sir2p-containing fractions are then added to the equilibrated calmodulin sepharose beads and the binding of Sir2p to the beads is allowed to proceed at 4 for 1 h with constant rotation. The beads are then packed into an econo-pac disposable plastic column and washed with 50 ml Binding Buffer A. Elution of the calmodulin bound fraction is carried out using
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Buffer B and 1 ml fractions are collected. Remove 50 l for protein estimation and protein blotting. Gel Filtration Chromatography
Gel filtration chromatography, which is commonly referred to as size exclusion chromatography, is a method for the separation of molecules on the basis of their size and shape. We chose Superose 6 resin because it can withstand higher back-pressure and has low ionic or hydrophobic interactions therefore reducing loss of protein during the purification. The gel filtration chromatography also allows us to change buffers in the sample. We initially checked the apparent molecular weight of Sir2p-containing complexes by applying yeast whole cell lysates, prepared from logarithmically growing cells directly on a Superose 6B column. Column fractions were analyzed by SDS-PAGE followed by immunoblotting using antiserum specific to Sir2p. This analysis indicated that Sir2p eluted from the column in two distinct peaks of apparent molecular weight of 900 and 70 kDa. The 70 kDa is most likely free Sir2p, which has a predicted molecular weight of 63 kDa, while the 900 kDa peak represents the major Sir2p-containing complexes. This analysis was also done initially to determine the stability of the Sir2p complexes under various conditions. A partially purified fraction of the Sir2p-containing complexes was treated with DNAse I, RNAse A, or high concentrations of KCl (500 mM) for 30 min at room temperature and was then subjected to gel filtration analysis. Neither nuclease treatment nor high salt concentration affected the overall size of the complex. Solutions Buffer A 50 mM HEPES 7.4 150 mM KCl 12.5 mM MgCl2 0.1% NP-40 10% Glycerol All the protease inhibitors. Materials Prepacked Superose 6 column (30 ml) HR 10/50 (Amersham Pharmacia).
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Method A Superose 6 column (30 ml size) was equilibrated with 150 ml of buffer A at a flow rate that did not exceed 50 cm/h. Use caution while connecting the column to avoid introducing any air bubbles. Wash the column with 3 volumes of buffer A to equilibrate the resin. Load 2 ml of the Sir2p containing fractions onto the Superose 6 column and collect 1 ml fractions after loading is complete. Remove 100 l of each fraction for protein blotting and Western analysis. These conditions were maintained for all our analysis for ease of comparison across experiments. The Sir2p complex is usually observed in fractions 7–9. It is important to calibrate the gel filtration column by running a set of standard molecular weight marker proteins through the column. Ensure the same volume as sample is also loaded in this case. Red Sepharose Chromatography
Sir2p is an NAD dependant histone deacetylase. We took advantage of this to further purify Sir2p containing complexes using a Red Sepharose CL 6B matrix that has a ligand mimic of NAD, Procion Red HE-3B coupled to it. We used this procedure to further purify and concentrate the sample. Solutions Buffer A 50 mM HEPES pH 7.0 12.5 MgCl2 10% Glycerol 0.1% NP-40 All protease inhibitors. Buffer B 50 mM HEPES pH 7.0 12.5 MgCl2 10% Glycerol 0.1% NP-40 1 M KCl All protease inhibitors. Materials HR 16 column (Amersham Pharmacia).
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Method An 8-ml HR 16 column is packed with the Red Sepharose matrix equilibrated as described for the SP-Sepharose resin. The column is equilibrated in Buffer A with 100 mM KCl. The Sir2p containing fractions are loaded on to this column. The column is then washed with 5 column volumes of 300 mM KCl in buffer A. The bound protein is eluted with 0.1% SDS, 2 ml fractions are collected and analysed by Western blots. We have not been successful in eluting the Sir2p containing complex from this column in its native form. We have also observed that Sir2p binds a Phenyl-Sepharose column with very high affinity in buffer containing 1.0 M KCl but we have once again only been able to elute these complexes from the Phenyl-Sepharose column following denaturation with 0.1% SDS. Purification of Recombinant Sir2p from E. coli
Solutions Lysis Buffer 50 mM HEPES pH 7.0 150 mM NaCl 0.1% Tween 20 20 M Zinc acetate All the protease inhibitors. Wash Buffer 50 mM HEPES pH 7.0 150 mM NaCl 10% Glycerol 10 mM Imidazole HCl All protease inhibitors. Elution Buffer 50 mM HEPES pH 7.0 150 mM NaCl 10% Glycerol 150 mM Imidazole HCl All protease inhibitors. Dialysis Buffer 25 mM HEPES, K+, pH 7.6
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12.5 mM MgCl2 0.1 mM EDTA 10% (v/v) Glycerol 0.1 M KCl 0.1% Tween 20. Materials 20 ml of BD Talon‘ resin (Clonetech) Econo-pac disposable chromatography column (Biorad). Method Transform E. coli DE81 pLysS cells with plasmid pRO 655. The Sir2 coding region is cloned in frame with the T7 6 His tags in a pET28 vector. Select transformants on kanamycin plates. Grow 1 L of cells to an A600 ¼ 0.6 in LB þ Kan. Add IPTG (1 mM final) to induce expression of the recombinant protein and grow the cells for a further 2.5 h. The cells are pelleted by centrifugation at 5000 rpm for 5 min at 4 using a Sorvall GS-3 rotor. The cells are washed in 1 L 20 mM Tris–HCl pH 7.8 and the cell pellets are frozen at 80 . The cell pellets are resuspended in 25 ml of lysis buffer and the A600 of the suspension is measured (10 l in 1 ml water). Sonicate the cell slurry for 2.5 min (30 s 5 pulses). Remeasure A600 (10 l in 1 ml) and the absorbance of the suspension should be approximately 25% of the original. Spin the lysed cell extracts in a Sorval SS34 rotor at 12,000 rpm for 20 min at 4 . The supernatant should contain most of the recombinant Sir2p. The Sir2p containing extract is added to 1.5 ml equilibrated settled BD Talon‘ resin in a 50 ml falcon tube (equilibrated in 50 mM HEPES pH 7.0, 150 mM NaCl, 10% glycerol, 0.5% Tween 20). Rotate the extract for 60 min at 4 on a rotator. Pour this slurry into a disposable Biorad Econopac column attached to a fraction collector. Connect a reservoir of wash buffer to the column and wash the column with 15 ml of Wash Buffer (10 column volumes) collecting fractions. Switch the reservoir from the wash buffer to the elution buffer and continue to collect fractions. Analyze 20 l of all collected fractions by SDS polyacrylamide gels. Dialyze appropriate fractions against dialysis buffer for 3 h at 4 and freeze in aliquots at 80 .